Common fragile sites are regions prone to gaps and breaks on metaphase chromosomes when DNA replication is perturbed. These sites, which span hundreds of kilobases, do not arise from mutation but are a normal component of chromosome structure. Over the past decade, we and others have shown that common fragile sites are "hot spots" for translocations, deletions, sister chromatid exchanges and integration of transfected DNA in cultured cells. With the cloning of fragile site loci, several lines of investigation have shown that common fragile sites display the same characteristics in many tumor cells, in vivo, and have strengthened a long-held hypothesis that common fragile sites are involved in genome rearrangements in cancer. Yet, little is known about the molecular mechanisms responsible for instability at fragile sites. We have recently identified the major molecular pathways affecting fragile site stability. These are the intra-S and G2/M checkpoint pathways in which the ATR and BRCA1 genes play central roles. From this, we hypothesize that fragile sites are unreplicated regions that normally activate the S/G2-M checkpoint and repair pathways, and that fragile sites and associated rearrangements in tumor cells are "signatures" of stalled replication forks. We will build on these important new findings and others made over the past decade to further address the question of what molecular mechanisms are responsible for fragile site maintenance and instability and what components of chromosome structure are responsible for this instability. Five aims are proposed to address these questions: (1) to further elucidate the role of genes involved in the S-phase and G2/M checkpoint pathways on fragile site maintenance and instability; (2) to examine the role of these genes in the induction of fragile sites by additional biologically-relevant agents and in induction of the fragile X site; (3) to investigate the role of genes recently implicated in the repair of stalled replication forks in fragile site stability and repair; (4) to determine the sequences or chromatin structure responsible for fragile site instability; and (5) to determine replication timing, the spacing of replication origins, replication fork velocity and timing of origin firing at fragile sites, using conventional and new molecular combing approaches. From these studies, we will further elucidate the specific molecular mechanisms underlying fragile site maintenance, instability and repair, and provide new insights into the function of genes critical to checkpoint control and DNA repair in response to stalled replication forks and their effects on human chromosome integrity. [unreadable] [unreadable]